CN109937092B

CN109937092B - Microfluidic chip with microbead integration system and method for integrating receptors in chip

Info

Publication number: CN109937092B
Application number: CN201780069958.8A
Authority: CN
Inventors: Y.特米兹; M.利姆; E.德拉马切
Original assignee: International Business Machines Corp
Current assignee: International Business Machines Corp
Priority date: 2016-11-03
Filing date: 2017-10-19
Publication date: 2021-07-30
Anticipated expiration: 2037-10-19
Also published as: GB2570596A; JP2020501115A; JP6924263B2; CN109937092A; DE112017004280T5; US9770717B1; DE112017004280B4; WO2018083563A1; GB201905496D0; GB2570596B

Abstract

The present invention is particularly directed to microfluidic chips. The chip includes a main microfluidic channel on one side of the chip, and a bead integration system. The bead integrated system is disposed on the one side of the chip. It includes a secondary microfluidic channel transverse to and in flow communication with the primary microfluidic channel so as to form an intersection therewith. The intersection is delimited by structured elements arranged in the main microfluidic channel. The structured elements are configured to retain, at the intersection, beads flowing in a bead suspension that advances in the auxiliary microfluidic channel and through the intersection. Further, such structured elements are configured to allow liquid advancing in the main microfluidic channel to pass through the intersection by passing through the structured element. The present invention is also directed to related apparatus and methods.

Description

Microfluidic chip with microbead integration system and method for integrating receptors in chip

Technical Field

The present invention generally relates to the following fields: microfluidics, microfluidic chips, and devices and methods for integrating receptors into microfluidic devices.

Background

Microfluidics deals with the behavior, precise control and manipulation of small volumes of liquid, typically confined to channels on the micrometer length scale and to volumes typically in the sub-milliliter range. The salient features of microfluidics arise from the unique behavior exhibited by liquids at the micron length scale. The flow of liquid in a microfluidic is typically laminar. Volumes below one nanoliter can be achieved by fabricating structures with lateral dimensions in the micrometer range. Reactions (by diffusion of reactants) limited by large size can be accelerated. Finally, parallel flow of liquids can be accurately and reproducibly controlled to allow chemical reactions and gradients to form at liquid/liquid and liquid/solid interfaces.

Microfluidic devices generally refer to microfabricated devices used for pumping, sampling, mixing, analyzing, and metering liquids. Instead of using active pumping means, microfluidic devices are known which use capillary forces to move a liquid sample within the microfluidic device. This makes the device easier to operate and less expensive, since no integrated or external (active) pump is required. However, particles, contaminants, and other problems during manufacturing can compromise capillary-based filling of the device.

Point-of-care diagnostic microfluidic devices are devices meant to be used by non-technical personnel at the patient's side or in the field and possibly at home. Existing point-of-care devices typically require loading a sample into the device and waiting a predefined time until a signal (usually an optical or fluorescent signal) can be read. The signal originates from a (bio) chemical reaction and relates to the concentration of the analyte in the sample. These reactions can be time consuming and difficult to achieve because they require optimal timing, flow conditions for the sample, and accurate dissolution of the reagents in the device. The reaction typically comprises a fragile reagent such as an antibody. Air bubbles may be generated in the device which may cause the test to fail. In addition, debris in the device can block liquid flow. In devices where the liquid must be split in parallel flow paths, filling may not occur at the same flow rate, and this may skew or invalidate the test.

In many analytical devices, with regard to the detection of analytes, in order to bind and accumulate the analyte, it is necessary to localize receptors in the region of the device. The localization of receptors is a challenging problem, especially for devices manufactured in large numbers at reasonable cost. In particular, when it is desired to close the assay device, it is sometimes difficult to introduce receptors in the area of the device. For capillary active devices, an additional difficulty is controlling the flow of receptor-containing solutions and avoiding diffusion of such solutions.

The positioning of the receptor can be done using photolithography. However, such techniques are expensive, slow, and may lack flexibility and compatibility with fragile receptors such as antibodies. Spotting (spotting) may also be used (e.g. inkjet, needle or brush-pin spotting). However, such techniques result in spreading of the liquid, drying artifacts, aggregation and uneven distribution of the receptor. Another technique commonly used is to topically dispense a solution containing the receptor on a porous medium such as paper or cellulose. However, this leads to a lack of resolution and uneven receptor density, which hinders multiplexing, miniaturization and signal quantification. Therefore, a solution is needed that makes it possible to easily integrate receptor microbeads in an assay device.

Disclosure of Invention

According to a first aspect, the invention is embodied as a microfluidic chip. The chip includes a main microfluidic channel on one side of the chip, and a bead integration system. The bead integrated systems are arranged on the same side of the chip. It includes a secondary microfluidic channel transverse to and in flow communication with the primary microfluidic channel so as to form an intersection therewith. The intersection is delimited by structured elements arranged in the main microfluidic channel. The structured elements are configured to retain, at the intersection, beads flowing in a bead suspension that advances in the auxiliary microfluidic channel and through the intersection. Further, such structured elements are configured to allow liquid advancing in the main microfluidic channel to pass through the intersection by passing through the structured element.

The above solution makes it possible to slow down and speed up the integration of microbeads, which typically comprise receptors. For example, the above-described device and correspondingly the integrated method do not require centrifugation to load beads or pellets, which is a time-consuming step. The beads can be loaded at a distance from the primary channel without having to locally dispense the beads directly in the primary channel. Bead integration can therefore be achieved simply and quickly, for example within a few minutes, and can be unattended.

In an embodiment, the structured elements comprise protruding elements protruding from the lower wall of the main microfluidic channel. Such elements may be, for example, pillars that are easily patterned.

The protruding elements may extend along two parallel lines across the main microfluidic channel, which lines partially delimit the junction. According to one embodiment, the protruding elements are spaced apart from each other so as to form openings for the passage of liquid therethrough.

For example, the protruding elements have an average diameter between 4 and 18 μm, the average gap between two consecutive protruding elements in each of the two parallel lines is between 2 and 8 μm, the two parallel lines being spaced at an average distance between 12 and 50 μm.

In one embodiment, the main microfluidic channel includes a lateral, moisture-resistant capillary structure formed at an edge sidewall of the main microfluidic channel adjacent the intersection. This may reduce lateral diffusion of the liquid (from the primary and/or secondary channels) and slow the progression of the liquid in the primary channel.

In an embodiment, the chip further comprises: a sample loading area in flow communication with the main microfluidic channel on one side of the intersection; and a capillary pump in flow communication with the main microfluidic channel on the other side of the junction. The main microfluidic channel connects the sample loading area to the capillary pump, thereby defining a liquid flow direction D (extending from the sample loading area to the capillary pump). Analysis of the liquid may be done in the main channel (or in the shunt channel) after the liquid has interacted with receptors on, for example, microbeads captured at the intersection.

The chip may contain two types of secondary microfluidic channels (i.e., first and second secondary microfluidic channels), each located on one side of the main channel. The bead integrated system may, for example, comprise a bead suspension loading region on one side of the main microfluidic channel and in flow communication with the main microfluidic channel via the first auxiliary microfluidic channel. The bead integration system may further comprise one or more second auxiliary microfluidic channels on the other side of the main microfluidic channel and in flow communication with the intersection. The one or more second auxiliary microfluidic channels may drain liquid passing through the intersection laterally from the bead suspension, rather than through the main channel, and therefore do not impede analysis of the analyte solution.

In embodiments, the bead integration system may further comprise an auxiliary capillary pump on the other side of the main microfluidic channel and in flow communication with the intersection via one or more second auxiliary microfluidic channels. The auxiliary pump helps to pump liquid from the bead suspension that has passed through the intersection.

In one embodiment, the first auxiliary microfluidic channel has a first opening to the intersection and the one or more second auxiliary microfluidic channels correspondingly have one or more second openings to the intersection. One or more second openings are provided in the side wall of the main microfluidic channel at the level of the intersection. Each of the one or more second openings may be sized to prevent the beads from exiting the intersection and re-entering the one or more second auxiliary microfluidic channels.

In an embodiment, the first auxiliary microfluidic channel extends substantially perpendicular to a portion of the main microfluidic channel at the level of the intersection. This makes it possible to maximize the distance from the bead suspension loading pad to the intersection (everything is otherwise the same) to avoid contaminating the main channel.

In an embodiment, on one side of the chip, the bead suspension loading area is at least partially surrounded by a moisture-resistant structure arranged at the periphery of the bead suspension loading area. This prevents spreading of the droplets when loading the bead suspension.

In an embodiment, the auxiliary microfluidic channel is in flow connection with the junction via a tapered portion widening towards the junction. This mitigates the risk of microbeads blocking at the entrance of the intersection or flowing back into the auxiliary channel towards the microbead suspension loading area when integrating microbeads.

In one embodiment, the main microfluidic channel (in a given liquid flow direction D extending from a liquid loading point in the main microfluidic channel to the junction) exhibits successively: a constriction and a tapered portion that widens towards the intersection. These additional structures help maintain stable fluid flow through the intersection, although the structured elements delimit the intersection (which must slow the liquid progression near the intersection).

In an embodiment, the bead integrated system further comprises a plurality of auxiliary microfluidic channels. Each of the auxiliary microfluidic channels intersects the main microfluidic channel on one side of the main microfluidic channel and is in flow communication with the main microfluidic channel so as to form a respective intersection therewith. Each of the intersections is delimited by a structured element arranged in the main microfluidic channel. Following the same principle as previously described, the structuring element is configured to retain, at each intersection, a bead flowing in a bead suspension that advances in and through the respective auxiliary microfluidic channel. The structured elements may also allow liquid advancing in the main microfluidic channel to pass through each intersection through the structured elements delimiting the intersection. Having multiple auxiliary microfluidic channels allows for multiplexing.

In one embodiment, the plurality of auxiliary microfluidic channels and corresponding intersections may also be desirable to simply widen the total area spanned by the intersections (and thus gather more beads) while maintaining control over bead distribution. For example, two adjacent intersections may be partially delimited by a single line of structured elements (i.e. elements protruding from the lower wall of the main microfluidic channel).

In embodiments relating to multiplexing, the intersections are distant, i.e. two consecutive intersections may be partially delimited by respective pairs of parallel lines of structured elements (again protruding from the lower wall of the main microfluidic channel), such that each of the pairs of parallel lines of structured elements partially delimits one of the intersections.

The present microfluidic chip may be provided with microbeads trapped therein at the intersection(s). In an embodiment, the captured microbeads substantially form a monolayer of microbeads. To that end, the primary microfluidic channel and the secondary channel may have substantially the same depth, which is less than twice the average diameter of the microbeads. In an embodiment, the chip is partially encapsulated with a film covering the intersection. The film may be, for example, a dry film resist, which may simply be laminated on top of the chip to seal the top of the chip.

According to other aspects, the invention is embodied as a method of integrating a receptor in a microfluidic chip according to embodiments as discussed herein. The method essentially comprises: loading a bead suspension in the auxiliary microfluidic channel for the bead suspension to advance in the auxiliary microfluidic channel and through the intersection such that the beads in the bead suspension are captured at the intersection. The microbead comprises a receptor.

In an embodiment, the method further comprises partially sealing the chip with a film covering the intersection. As previously described, in the embodiment, the film is a dry film resist, which is laminated to a portion of the sealing chip.

According to a final aspect, the invention is embodied as a method of using a microfluidic chip according to the embodiments discussed above, wherein the captured microbeads include a receptor. The method includes loading a liquid including an assay liquid in the main microfluidic channel, advancing the liquid along the main microfluidic channel, through the intersection, and interacting there with receptors for the captured microbeads.

An apparatus and method embodying the invention will now be described by way of non-limiting example and with reference to the accompanying drawings.

Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

fig. 1 is a top view of a microfluidic chip according to an embodiment;

FIGS. 2 and 3 are top views of similar devices as in the examples, showing bead integration (FIG. 2) and interaction of analyte liquid with the integrated beads (FIG. 3);

FIG. 4 is a 3D view of the device of FIG. 1, focusing on the intersection between the secondary microfluidic channel and the primary microfluidic channel of the chip where microbeads are captured, according to one embodiment;

FIG. 5 shows steps of manufacturing a sealed chip according to an embodiment;

fig. 6 is an experimental image of a top view of a microfluidic chip with two intersections of captured microbeads according to an embodiment;

fig. 7 shows an experimental image of a top view of another microfluidic chip. The magnified image shows structural details about the intersection between the secondary microfluidic channel and the primary channel of the chip in an embodiment;

FIG. 8 is an experimental image of a top view of another microfluidic chip designed for multiplexing in the examples;

FIG. 9a shows one of various possible designs of a microfluidic chip according to other embodiments (top view);

FIG. 9b shows one of various possible designs of a microfluidic chip according to other embodiments (top view);

FIG. 9c shows one of various possible designs of microfluidic chips (top view) according to other embodiments;

FIG. 9d shows one of various possible designs of microfluidic chips (top view) according to other embodiments;

fig. 9e shows one of various possible designs of a microfluidic chip according to other embodiments (top view);

FIG. 9f shows one of various possible designs of microfluidic chips (top view) according to other embodiments;

FIG. 9g shows one of various possible designs of a microfluidic chip (top view) according to other embodiments; and

fig. 9h shows one of various possible designs of a microfluidic chip according to other embodiments (top view).

Detailed Description

The figures show simplified representations of devices or parts thereof as comprised in the embodiments. The features depicted in the drawings are not necessarily to scale. Elements that are the same or functionally similar in the drawings have been assigned the same reference numerals, unless otherwise indicated.

The following description is made as follows. First, a general embodiment and a high-level modification (first section) are described. The next section sets forth more specific embodiments and technical implementation details (second section).

1. General embodiments and high level variants

With reference to fig. 1-4, embodiments of the present invention are first described with respect to a microfluidic chip 1. The chip 1 basically comprises a main microfluidic channel 12 and a bead integration system 20. The main microfluidic channel 12 is on one side of the chip 1. The bead integrated systems 20 are arranged on the same side of the chip 1.

The bead integration system 20 includes, inter alia, an auxiliary microfluidic channel 22. As discussed in detail later, it may actually include a plurality of secondary channels on one or each side of the primary channel 12. As shown in fig. 1-3, the secondary channel 22 is disposed transverse (e.g., perpendicular) to and in fluid communication with the primary channel 12 so as to form a junction 28. The

channels

12, 22, 23 are all in a plane on the same side of the device 1.

Intersection 28 is delimited by structured elements 26 arranged in main microfluidic channel 12. The structured elements 26 have two functions. First, they are configured to retain beads at intersection 28. In other words, when the microbeads 55 are introduced into the auxiliary passage 22 in the microbead suspension 50, the liquid advances toward the intersection 28 in the auxiliary passage 22 and then passes through the intersection 28. Beads that reach junction 28 are captured there as they remain at junction 28, and excess liquid 50 may be drained via primary channel 12 or via one or more other secondary channels 23 on the other side of the primary channel. Furthermore, the structured element 26 is configured so as to allow analyte liquid 60 advancing in the main microfluidic channel 12 to pass through the structured element 26 to and through the intersection 28 so as to interact with, for example, receptors on captured microbeads.

The present solution makes it possible to slow down and speed up the integration of microbeads 55 in device 1. They also make it unnecessary to resort to topical dispensing methods, for example methods of topically dispensing a solution containing the receptor on a porous medium such as paper or cellulose. Such methods mainly result in lack of resolution and uneven receptor density, which hinders multiplexing, miniaturization and signal quantification. Furthermore, the present device and method do not require centrifugation to pack (pack) microbeads (as is conventionally used in analytical devices) or sedimentation (as is commonly used in chromatography columns to pack microbeads), which operations are time consuming. In contrast, the present methods typically allow bead integration within minutes (even if not seconds).

The embodiments disclosed herein also avoid the following problems as encountered in prior art solutions: such as spreading of the liquid, drying artifacts, clustering of the receptors and uneven distribution. That is, the present method allows for clean integration of microbeads.

As shown, for example, in fig. 1, microfluidic chip 1 typically includes a sample loading region 11 in flow communication with main channel 12 on one side of intersection 28 to mitigate introduction of liquid sample (analyte) into chip 1. The capillary pump 13 will in embodiments be provided in flow communication with the main channel 12 on the other side of the junction 28. The main microfluidic channel 12 connects the sample loading area 11 to the capillary pump 13 via the intersection 28 accordingly, which allows a fully passive operation of the chip 1. No external pump is required, which makes the present device 1 capable of point-of-care diagnostics. As shown in fig. 1, the liquid flow direction D of the liquid sample 60 in the main channel 12 extends from the sample loading area 11 to the capillary pump 13. Each of the designs of fig. 1-9 assumes that the liquid flow direction D is along the x-axis.

In an embodiment, structured elements 26 comprise protruding elements. Such elements 26 in the embodiment protrude from the lower wall 12L of the main microfluidic channel 12, but they may also protrude from the upper seal or cover. However, providing such elements 26 directly on the main channel 12 makes it easier to assemble and obtain accurate placement of these elements near the intersection 28. Such elements 26 may be shaped, for example, as pillars as envisioned in fig. 4, as the pillars are relatively simple objects that are patterned. However, other structures 26 having openings or holes are contemplated to facilitate passage of the liquid 60 therethrough. In a variant, the structure (possibly patterned) formed by the rough surface in the vicinity of the region 28 may serve the same purpose. However, it is preferred to use a clean, protruding structure 26. Such structures are patterned, inter alia, using photolithography, direct laser writing, 3D printing, or replication methods based on hot stamping and injection molding techniques.

As further shown in fig. 1-4, the protruding elements 26 in an embodiment extend along two parallel lines across the main microfluidic channel 12. The intersection 28 is partially bounded laterally (along the y-axis) by the line drawn by the element 26. As implied by the present geometry, other structural elements help to delimit the intersection 28, such as the sidewalls 121 of the main channel 12. The projecting elements 26 are spaced apart from each other so as to form openings (holes) and allow the passage of the liquid 60 therethrough.

In terms of size, the protruding elements 26 need to be sized to conform to the desired area of the beads 55 and intersection 28. It also depends on the number of microbeads (and hence the number of receptors) and the flow rate and concentration of analyte 60 required for the test to be performed by the device 1. The main channel 12 needs to be dimensioned accordingly. As one will appreciate, the sizes of the primary

structured elements

12, 22, 26, 28 are interrelated and may require joint optimization, which may be accomplished using a trial and error approach. For example, using microbeads typically having an average diameter of 10 μm, the average diameter of the protruding elements 26 may be between 4 and 18 μm, for example 8 μm, so that they act as sufficiently robust pens and retain the microbeads. Meanwhile, in each of the two parallel lines formed by elements 26, the average gap (along the y-axis) between two consecutive elements 26 (e.g., as measured between the nearest peripheral vertices of two consecutive elements 26) may typically be between 2 and 8 μm, for example 4 μm. For example, two parallel lines separated by an average distance of between 12 and 50 μm (e.g., 25 μm) to allow one or more columns of microbeads to aggregate at intersection 28.

Referring now to fig. 1, 6 and 7, an embodiment of a microfluidic chip 1 comprises channels 12 that are laterally structured at the level of intersections 28. That is, main microfluidic channel 12 includes a lateral, moisture-resistant capillary structure 14 formed at an edge sidewall 121 of main channel 12 adjacent intersection 28. As best shown in fig. 6 and 7, the moisture-resistant capillary structures 14 may be patterned, for example, as a transverse grid of teeth or saw-tooth like structures 14. The lateral structures 14 need to be appropriately sized and spaced so as to exhibit an appropriate angle to drive the aqueous liquid by capillary action. This makes it possible to reduce lateral spreading of the

liquids

50 and 60 near the intersection 28.

In embodiments the bead suspension 50 should of course not diffuse too fast in the main channel 12, since this main channel 12 is normally used for analysis; the liquid 50 does hinder such analysis. Furthermore, as the analyte 60 fills the channel 12, the anti-wetting capillary structure 14 decelerates the liquid meniscus 61, i.e. it inhibits lateral progression of the meniscus 61 and mitigates the risk of asymmetric filling of the channel 12 (and thus the risk of bubble formation). For example, if salt crystals remain in the channels 12 after drying (at the time of manufacture), such crystals may accelerate filling in the main channels 12 because they are polar. In practice, salt crystals may accumulate in the lateral corners of the main channel 12. A similar effect may be obtained by chemically treating the side surfaces of the tunnel 12 near the intersection 28. However, patterning the lateral, moisture-resistant structure 14 is simpler from a manufacturing standpoint.

As previously mentioned, an embodiment of the microfluidic chip 1 comprises a plurality of auxiliary channels 22, 23(23 a-c). The auxiliary microfluidic channel 22 may for example be referred to as a first auxiliary channel 22 (or channel section). As shown in fig. 4, 6 and 7, the bead integrated system 20 may further comprise a bead suspension loading region 21 on one side of the main microfluidic channel 12 and in flow communication therewith, i.e. via the auxiliary channel 22. This facilitates the introduction of the bead suspension 50, which can be done at a safe distance from the main channel 12 in order to prevent spreading of the liquid and drying artifacts. In this regard, the auxiliary aisle 22 in the embodiment extends perpendicular to the main aisle 12 at the level of the intersection 28 in order to maximize the distance from the loading area 21 to the intersection 28 (all otherwise identical).

There may be several channel portions 22, 23. The bead integration system 20 may include, inter alia, one or more second auxiliary microfluidic channels 23, 23a-c on the other side of the main channel 12 in flow communication with the junction 28. The second auxiliary microfluidic channels 23, 23a-c may drain liquid 50 laterally from the bead suspension as it passes through junction 28, rather than through main channel 12, and therefore do not impede analysis of the analyte liquid. For multiplexing purposes, multiple first auxiliary channels 22 may be required (as discussed later with reference to fig. 8) or simply widening the bead suspension inlet (as in fig. 9 h). Having several second auxiliary channels 23a-c for each first auxiliary channel 22 allows reducing the width of the channels 23a-c (and prevents microbeads from entering such channels 23 a-c).

As further shown in fig. 1-3, the microbead integrated system 20 in the embodiment includes an auxiliary capillary pump 24 opposite the microbead suspension loading area 21 with respect to the main channel 12. Auxiliary capillary pump 24 is in flow communication with junction 28 via one or more second auxiliary channels 23, 23 a-c. That is, the auxiliary microfluidic channel 22 connects the bead suspension loading area 21 (e.g., fluid loading pad) to the main channel 12 at a junction 28, and the auxiliary capillary pump 24 is fluidly connected to the junction 28 via one or more second auxiliary channels 23, 23 a-c.

Again, auxiliary capillary pump 24 allows for a passive system, i.e., advancement of bead suspension 50 is passively driven by capillary pump 24 on the other side of intersection 28. Best results are obtained if the main channel 12 and the auxiliary channels 22, 23 all have the same depth with respect to their junction 28, which further simplifies the manufacturing process. This also helps to control the number of beads within the intersection. Still, the (wet)

channels

12, 22, 23 also function as passive capillary pumps.

Due to the

passive capillary members

12, 22, 23, 24, the present device allows unattended bead integration. For example, after injecting a suspension of microbeads into the microbead integrated system 20, the microbeads will self-assemble at the intersection 28, and the suspension will gradually evaporate. This allows for very efficient integration of microbeads and fabrication of microfluidic devices on a batch level. In other words, parallel bead integration can be achieved using multiple devices placed on a tray (tray) or using roll to roll manufacturing techniques. Drying of the liquid 50 may also be accomplished using an oven and controlled ambient conditions (temperature and relative humidity) to balance both the rate of evaporation of excess liquid and the rate of loading of the intersection 28 with microbeads.

One may want to obtain a monolayer of crystallized microbeads 55 at intersection(s) 28 to better control the actual number of microbeads (and thus the number of receptors) integrated. According to experiments conducted by the present inventors, monolayers of microbeads can be very easily obtained by main channels 12 having a depth of 5 μm and a width of 100 μm, and the space between parallel lines of structuring element 26 is between 10 and 25 μm using a 0.2% solution of 4.5 μm microbeads. Further, to help achieve this goal, the lower wall 12L of the main channel 12 may be patterned at the level of the intersection 28 so as to exhibit bead retention features (e.g., an array of bead capture wells). This is discussed again in section 2.2.

In the embodiments illustrated by fig. 4, 6 and 7, one or more second auxiliary microfluidic channels 23, 23a-c each have one or more second openings 23oi to intersection 28. A second opening 23o is provided in the side wall 121 of the main channel 12 at the level of the intersection 28. The first auxiliary microfluidic channel 22 has a first opening 22t to a junction 28 formed with the main channel 12. Each of the one or more second openings 23o is narrower than the first opening 22t (as measured in a direction parallel to the liquid flow direction D, i.e. along the x-axis). The second opening (and thus the second auxiliary channel) may be sized to prevent beads from leaving the intersection and entering the second auxiliary channel.

As further shown in fig. 4, 6 and 7, the auxiliary microfluidic channel 22 in an embodiment is connected to the junction 28 via a tapered portion 22t (which widens towards the junction 28). This alleviates the risk of the microbeads 55 blocking at the entrance of the intersection 28 or flowing back into the auxiliary channel 22 toward the liquid loading area 21 when integrating the microbeads.

As best shown in fig. 7, in an embodiment, the main channel 12 shows a constriction 15 and a conical portion 16 in series. I.e. the tapered portion 16 is patterned directly after the constriction 15. The tapered portion 16 widens towards the intersection 28. As the inventors have realized, while the structured elements 26 delimit the junction 28, which necessarily would interfere with the progression of the liquid 60 in the main channel, the continuous

lateral structures

15, 16 help to maintain a steady flow of liquid through the junction 28. Similar lateral structures can also be seen in fig. 9a-e and 9 h. If necessary, a series of several converging conical pairs may be provided along the liquid flow direction D (from the liquid loading zone 11 to the junction 28).

Typical dimensions of the

lateral structures

15, 16 range from 2 μm to 50 μm. In the case of a structure 50 μm large, the main channel may be 200 μm wide. However, more important than the dimensions of

structures

15 and 16 is the angle formed by such structures. By forming an angle less than or equal to 90 degrees, the

structures

15, 16 will form a capillary barrier to prevent liquid from advancing toward the intersection 28. In other words,

structures

15, 16 will serve as obstacle (ping) sites. An angle of about 45 degrees results in a more aggressive obstacle point than an angle between 45 and 90 degrees. Smaller angles may also impede liquids, but these may present considerable manufacturing challenges, particularly if methods other than photolithography are used.

Referring to fig. 6, 8 and 9h, the microbead integrated system 20 in the example includes a plurality of auxiliary channels 22. Each of the secondary channels 22 is transverse (e.g., perpendicular) to and in flow communication with the primary channel 12, the secondary channels 22 being on one side of the primary channel 12. Which together with the main channel 12 form a respective junction 28. Each intersection 28 is again delimited by a structured element 26 arranged in the main channel 12 so as to retain a bead 55 therein, while allowing sample liquid 60 to enter and pass through each intersection 28. As previously mentioned, this can serve two purposes: it allows multiplexing (as in fig. 8) or simply widening the liquid inlet across the main channel 12 (as in fig. 9 h).

Referring first to fig. 9h, two adjacent intersections 28 are partially delimited here by a single line of structured elements 26. As previously mentioned, each line of structured elements 26 may comprise elements protruding from lower wall 12L of main channel 12. In this way, adjacent intersections 28 are obtained which widen the total area available for bead aggregation. In a variant, one may simply widen the area spanned by a single intersection 28 (as delimited by parallel lines of the structure 26). However, this solution makes it more difficult to maintain control over the bead distribution as it collects at the intersection. Of course, one may want to maintain a certain ratio between the distance between parallel lines of structured elements 26 and the bead diameter (e.g., between 2: 1 and 3: 1) to achieve a desired bead distribution in region 28. Thus, if a wider area 28 is desired, one can do so by patterning several adjacent intersections 28 (e.g., separated by a column of columns 26).

In a variation as depicted in fig. 8, the intersections 28 are a distance from each other. In other words, two successive intersections 28 are now laterally delimited by respective pairs of parallel lines of the structured element 26. Again, this structuring element 26 is obtained in the embodiment as an element projecting from the lower wall 12L of the main channel 12. Thus, each pair of parallel lines of structured elements 26 laterally delimits a respective, single intersection 28. This design allows for well separated multiplexing.

Referring again to fig. 2-3 and 5, another embodiment of the invention will now be briefly discussed with respect to a method of integrating receptors in a microfluidic chip 1 as described above. Such an approach is quite simple due to the chip design considered herein. First, step S10 of fig. 5, provides an original microfluidic chip 1 (as e.g. fig. 1) in which microbeads have not yet been integrated. Then, S20 loads bead suspension 50 in auxiliary channel 22, for example, via loading pad 21. The beads 55 include receptors that can be tested later. The loaded bead suspension 50 then enters the auxiliary channel 22 towards the intersection 28 and passes through the intersection 28 (the liquid being expelled in the main channel or better in the opposite auxiliary channel 23, for example as facilitated by the capillary pump 24) so that the bead 55 is spontaneously captured at the intersection 28.

Thus, and as shown in fig. 5, 6, the present chip 1 may thus be equipped (as a final product prepared for example for testing purposes) with microbeads 55 laterally captured at the intersection(s) 28.

As further depicted in fig. 5, an embodiment of such a method further comprises S30 partially sealing the chip 1 with a cover or film 70 for covering the intersection (S) 28 in order to prevent microbeads from escaping the intersection 28 when, for example, handling, packaging or transporting the chip 1. The membrane 70 may cover, inter alia, the

channels

12, 22, 23, the capillary pumps 13, 24, and the loading pad 21. However, in an embodiment, the opening will make the liquid loading zone 11 accessible. Openings may be predefined in the film 70 and then laminated. In a variant, the film may comprise precut lines corresponding to the desired openings, for example on which labels are glued to ease the removal of the corresponding film portions. The user will simply remove the portion of the membrane corresponding to the opening to begin the test.

In an embodiment, one may want to obtain a captured microbead 55 that essentially forms a single layer of microbeads as envisioned in fig. 4. To achieve that goal, in an embodiment, the primary channels 12 and the secondary channels 22 will have the same depth, which is less than twice the average diameter of the microbeads (e.g., 10 μm), such as less than 20 μm.

Referring now to fig. 3, a final embodiment of the invention will now be described in relation to a method of using the microfluidic chip 1 as described herein. It is envisaged that the chip 1 comprises microbeads which have been integrated therein, for example according to the method described above. The captured microbeads 55 typically include a receptor for the analyte to react with. The user simply loads S40 a liquid sample 60 containing one or more types of analytes in main channel 12. The loaded liquid 60 then proceeds in and along the main microfluidic channel 12, through the intersection 28, to interact there with the receptors for the captured microbeads 55. The liquid 60 then remains at the junction(s) 28 and proceeds along the main channel 12, for example towards the capillary pump 13. Control and detection can be performed directly on the main channel or on the shunt channel according to known techniques (including microscopes, smartphones or electrodes), which need not be discussed in detail here.

The above-described embodiments have been described briefly with reference to the drawings, and may incorporate a plurality of variations. Several combinations of the above features are contemplated. An example is given in the next section.

2. Specific embodiment/technical implementation details

2.1 Point-of-care diagnostics, Mobile health and safety features

The present chip 1 embodiment comprises a test device for diagnostic tests, such as a so-called rapid test device or a rapid diagnostic test device. A Rapid Diagnostic Test (RDT) device is a device for rapid and easy medical diagnostic testing. They typically allow results to be obtained within a few hours. They include, inter alia, point of care (POC) testing devices and over-the-counter (OTC) tests.

Such a test device may especially be portable, e.g. a handheld device, such as e.g. a blood glucose meter, a dipstick or a test kit for detecting one or several analytes (e.g. C-reactive protein, cardiac markers, viral antigens, allergens, transgenic organisms, pesticides, contaminants, metabolites, cancer biomarkers such as carcinoembryonic antigens, etc., therapeutic drugs, drugs of abuse, etc.), or pregnancy or fertility tests. Such devices may also be used to detect cellular receptors or antibodies (as in the case of serum tests). In general, the solution can be applied to any receptor-ligand assay comprising a DNA-based assay. For example, the microbeads may be coated with DNA probes. Such probes can hybridize to complementary targets of DNA flowing in the main channel. Double stranded DNA with an inserted dye or a labeled DNA reporter strand may be used to reveal hybridization. More generally, the present device may be any type of RDT device (POC or OTC device). Furthermore, the test device may be used for conducting assays beyond medical diagnosis, e.g. for detecting toxins in water, etc. As the skilled person will appreciate, there are potentially many applications for such testing devices. Detection may be done using, for example, a low-end microscope or smartphone to enable mobile "health.

The device further comprises an optically readable medium, wherein the medium comprises a pattern of spots of material arranged on a surface of the device. The spots may especially be inkjet printed to ensure accurate placement of the spots and to ensure reasonable manufacturing time. Several patterns may be present at different locations on the device. The pattern thus formed may be human and/or machine readable. They may in particular encode security information (e.g. security keys) or be designed to reveal a pattern indicating whether the device has been used. More generally, the security pattern allows information to be encoded directly on the testing device and is therefore more difficult to counterfeit or forge, and the security pattern may therefore be useful for testing to detect counterfeiting or forgery or to signal spoofing tests (e.g. tests already in use).

In an embodiment, the test device further comprises a cover covering the speckle pattern, wherein the cover is light transmissive. The spots of material forming the pattern are thus located under the cover, which makes them difficult to replicate or imitate. The pattern (i.e. the key) may for example fit a 400 μm wide channel (whose width as a whole will be less than 1mm) structured in the SU-83010 surface or SU-83050 surface. The size of the spot is small enough to provide enough key elements. Only a few droplets are required per element, which leads to good optical contrast with few defects, thus leading to a key that is visible when imaging with a smartphone equipped with an external, inexpensive macro lens.

2.2 preparation of

The surface on which the main flow path 12 is formed is a surface of typically one of the following materials: a polymer (e.g., SU-8 polymer), silica, or glass. Other materials are contemplated, such as, for example, metal coatings. However, metal coatings may require more complex manufacturing methods (e.g., clean rooms or complex processes), or require toxic precursors.

Conventional manufacturing methods may be used to manufacture the present devices, including injection molding and hot stamping. 3D printing may also be used, but the

structures

15 and 16 may need to be slightly rounded in this case. However, one may want to use anisotropic dry etching techniques in embodiments to obtain bead-integrated

precision structures

14, 26.

Single step anisotropic dry etching of silicon (e.g., DRIE) can be used to advantage especially since they require only a single mask and provide high resolution patterning. In particular, a single step isotropic etch of silicon allows for an undercut and overhanging mask layer to be obtained to create partially closed bead-integrated trenches and channels.

One-step patterning of SU-8 can also be used, which also allows to obtain a reliable capillary valve. Both techniques can be mixed. That is, reliable valves, microfluidic channels and deep capillary pumps can be obtained due to SU-8 (which has a high volume capacity), while accurate bead-integrated structures can be obtained by DRIE.

For example, in an embodiment, the chip measures 19.5 x 9.4mm2 and includes

loading pads

11, 21,

microchannels

12, 22, 23 with electrodes embedded in the main channel 12 or the shunt channel (not shown), capillary pumps 13, 24, vents, cover films, and electrical contacts that mate with card edge sockets. Silicon substrates are used to take advantage of micromechanical processes and the good properties of Si and SiO2, such as channel etching with tapered sidewall profiles, hydrophilicity, thermal and chemical stability of capillary filled SiO2, mechanical robustness, compatibility of the SiO2 surface with many biomolecules, and well-defined and reliable chemical composition.

In the fabrication process, the channels are anisotropically etched in silicon using TMAH, and electrically passivated by thermal oxidation. The electrodes are patterned by metal evaporation and stripped after conformal coating and patterning of a single layer of photoresist. A short isotropic SiO2 etch was introduced prior to metal deposition to aid in stripping and recessing the electrodes. The lithographic parameters are optimized to achieve a minimum feature size of at least 5 μm in a trench 20 μm deep. Following the slicing and cleaning steps, a hydrophilic dry film cover was laminated at 45 ℃ to seal the microfluidic structure. SEM examination showed that the cover film was perfectly covered on the channels and capillary pumps. Due to the recessing step, the electrode shows minimal edge defects and a very flat surface appearance.

In a variation, the electrode is patterned on a flat Si surface with a SiO2 passivation layer using a metal lift-off or metal etch process. Additional processes, such as photolithographic patterning of SU-8 or dry film resist, are then used to pattern the microfluidic structure. Although not preferred, the electrodes may also be patterned on a cover substrate (or film) and then bonded to the substrate carrying the microfluidic structure using chip or wafer bonding techniques (e.g., film lamination, anodic bonding, direct bonding, thermoplastic bonding, adhesive bonding, etc.). In case there is already a chip function requiring electrodes (e.g. micro-heaters, electrophoretic or electrowetting electrodes, or galvanic, impedance, or electrochemical sensing electrodes, etc.), the electrodes for liquid monitoring as a whole may be patterned together with other electrode patterns or conductive layers.

The methods as described herein may be used in the manufacture of microfluidic devices, particularly wafer-based chips. The resulting chips can be distributed as bare die, for example, by a fabrication machine in raw wafer form (in other words, a single wafer with a plurality of unpackaged chips), or in packaged form. In the latter case, the chip is mounted in a single chip package (such as a plastic carrier) or a multi-chip package. In any case the chip can then be integrated with other chips or other microfluidic elements (tube ports, pumps, etc.), even if preferred for autonomous chip applications, as part of either (a) an intermediate product or (b) an end product.

While the invention has been described with reference to a limited number of embodiments, modifications and drawings, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In particular, features (like devices or like methods) recited in a given embodiment, variation or shown in the drawings may be combined with or substituted for other embodiments, variations or other features in the drawings without departing from the scope of the invention. Various combinations of the features described in relation to any of the above embodiments or variations are therefore contemplated and are within the scope of the appended claims. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, many other variations than those explicitly recited above are contemplated. For example, the microfluidic chip claimed herein may be fabricated as a microfluidic probe.

Claims

1. A microfluidic chip, comprising:

a main microfluidic channel on one side of the chip; and

a bead integration system disposed on the one side of the chip, the bead integration system comprising:

a first auxiliary microfluidic channel transverse to and in flow communication with the main microfluidic channel so as to constitute an intersection therewith, the first auxiliary microfluidic channel having a first opening to the intersection, the first auxiliary microfluidic channel being on one side of the microfluidic channel, and

one or more second auxiliary microfluidic channels in flow communication with the intersection, each of the one or more second auxiliary microfluidic channels having a second opening to the intersection, respectively, each of the second openings being provided in a sidewall of the main microfluidic channel, each of the second openings being narrower than the first opening, the one or more second auxiliary microfluidic channels being on the other side of the main microfluidic channel,

the intersection is bounded by a structured element disposed in the main microfluidic channel, the structured element configured to:

retaining microbeads flowing in a suspension of microbeads at the intersection, the suspension of microbeads progressing in the first auxiliary microfluidic channel and through the intersection to the second auxiliary microfluidic channel; and is

Passing a liquid advancing in the main microfluidic channel through the intersection.

2. The microfluidic chip according to claim 1, wherein:

the structured elements comprise protruding elements protruding from a lower wall of the main microfluidic channel.

3. The microfluidic chip according to claim 2, wherein:

the protruding elements extend only along two parallel lines across the main microfluidic channel, the lines partially delimiting the intersection, wherein the protruding elements are spaced apart from each other so as to form an opening for passage of liquid therethrough.

4. The microfluidic chip according to claim 3, wherein:

the protruding elements have an average diameter between 4 and 18 μm, the average gap between two consecutive protruding elements in each of the two parallel lines is between 2 and 8 μm, the two parallel lines are spaced apart by an average distance between 12 and 50 μm.

5. The microfluidic chip according to claim 1, wherein:

the main microfluidic channel includes a lateral, moisture-resistant capillary structure formed at an edge sidewall of the main microfluidic channel adjacent the intersection.

6. The microfluidic chip according to claim 1, wherein said chip further comprises:

a sample loading region in flow communication with the main microfluidic channel on one side of the intersection; and

a capillary pump in flow communication with the main microfluidic channel on the other side of the intersection, whereby the main microfluidic channel connects the sample loading area to the capillary pump, thereby defining a liquid flow direction D extending from the sample loading area to the capillary pump.

7. The microfluidic chip according to claim 1, wherein:

the microbead integrated system further comprises:

a bead suspension loading region on one side of the primary microfluidic channel and in flow communication with the primary microfluidic channel via the first auxiliary microfluidic channel.

8. The microfluidic chip according to claim 7, wherein said microbead integrated system further comprises:

an auxiliary capillary pump on the other side of the main microfluidic channel and in flow communication with the junction via the one or more second auxiliary microfluidic channels.

9. The microfluidic chip according to claim 7, wherein:

the first auxiliary microfluidic channel extends substantially perpendicular to a portion of the main microfluidic channel at the level of the intersection.

10. The microfluidic chip according to claim 7, wherein:

on the one side of the chip, the microbead suspension loading area is at least partially surrounded by a moisture-resistant structure disposed at a periphery of the microbead suspension loading area.

11. The microfluidic chip according to claim 1, wherein:

the microbead integrated system further comprises a plurality of first auxiliary microfluidic channels, each first auxiliary microfluidic channel transverse to and in flow communication with the main microfluidic channel on one side of the main microfluidic channel so as to form therewith a respective intersection, each of the intersections being bounded by structured elements arranged in the main microfluidic channel and configured to:

retaining, at each of the intersections, a bead flowing in a bead suspension that advances in a respective one of the first auxiliary microfluidic channels and through each of the intersections; and

passing a liquid advancing in the main microfluidic channel through each of the intersections through the structured element bounding it.

12. The microfluidic chip according to claim 11, wherein:

two adjacent intersections of the respective intersection are partially delimited by a single line of structured elements comprising elements protruding from a lower wall of the main microfluidic channel.

13. The microfluidic chip according to claim 11, wherein:

two successive intersections of the respective intersection are delimited by respective pairs of parallel lines of structured elements comprising elements protruding from the lower wall of the main microfluidic channel, such that each pair of parallel lines of structured elements partially delimit one of the intersections.

14. The microfluidic chip according to claim 1, wherein:

the chip also includes microbeads captured at the intersection.

15. The microfluidic chip according to claim 14, wherein:

the captured microbeads substantially form a monolayer of microbeads, the primary microfluidic channel and the secondary channel having the same depth that is less than twice the average diameter of the microbeads.

16. The microfluidic chip according to claim 14, wherein:

the chip is partially sealed with a film covering the intersection; and the film is a laminated dry film resist.

17. The microfluidic chip according to claim 1, wherein:

each of the plurality of second auxiliary microfluidic channels extends substantially perpendicular to a portion of the main microfluidic channel at the level of the intersection.

18. The microfluidic chip according to claim 17, wherein:

each of the second auxiliary microfluidic channels has a reduced width that prevents microbeads from entering the auxiliary microfluidic channel.

19. The microfluidic chip according to claim 3, wherein:

the second auxiliary microfluidic channel is between the two parallel lines.

20. A microfluidic chip, comprising:

a main microfluidic channel on one side of the chip; and

a first auxiliary microfluidic channel transverse to and in flow communication with the main microfluidic channel so as to constitute an intersection therewith;

one or more second auxiliary microfluidic channels in flow communication with the intersection, each of the one or more second auxiliary microfluidic channels having a second opening to the intersection, respectively, each of the second openings being provided in a sidewall of the main microfluidic channel, each of the second openings being narrower than the first opening, the one or more second auxiliary microfluidic channels being on the other side of the main microfluidic channel;

a bead suspension loading area on one side of the primary microfluidic channel and in flow communication with the primary microfluidic channel via the first auxiliary microfluidic channel, the first auxiliary microfluidic channel being between the bead suspension loading area and the intersection;

the first auxiliary microfluidic channel being in flow connection with the junction via a tapered portion widening towards the junction, the tapered portion being comprised in the first auxiliary microfluidic channel,

delimiting the intersection by a structure disposed in the main microfluidic channel that:

retaining beads flowing in a bead suspension at the intersection, the bead suspension advancing from the first auxiliary microfluidic channel to a tapered portion that widens toward the intersection, then reaching the intersection and passing through the intersection; and is

Passing liquid advancing in the main microfluidic channel through the intersection.

21. A microfluidic chip, comprising:

a main microfluidic channel on one side of the chip; and

a first auxiliary microfluidic channel transverse to and in flow communication with the main microfluidic channel so as to constitute an intersection therewith; and

one or more second auxiliary microfluidic channels in flow communication with the intersection, each of the one or more second auxiliary microfluidic channels having a second opening to the intersection, respectively, each of the second openings being provided in a sidewall of the main microfluidic channel, each of the second openings being narrower than the first opening, the one or more second auxiliary microfluidic channels being on the other side of the main microfluidic channel; and

a sample loading area in flow communication with the main microfluidic channel on one side of the intersection, the main microfluidic channel being between the sample loading area and the intersection; and

retaining beads flowing in a bead suspension at the intersection, the bead suspension advancing in the first auxiliary microfluidic channel and through the intersection; and is

Passing a liquid advancing in the main microfluidic channel through the intersection, wherein

In a direction extending from the sample loading area to the intersection, the main microfluidic channel successively exhibits: a constriction and a tapered portion, the tapered portion widening towards the intersection.

22. A method of integrating receptors in a microfluidic chip according to claim 1, the method comprising:

loading a bead suspension in the first auxiliary microfluidic channel for advancement of the bead suspension in the first auxiliary microfluidic channel and through the intersection such that beads in the bead suspension are captured at the intersection, wherein the beads comprise the receptor.

23. The method of claim 22, wherein the method further comprises:

partially sealing the chip with a film covering the intersection, wherein

Partially sealing the chip includes laminating the film, which is a dry film resist.

24. The method of claim 22, wherein:

the captured microbeads include a receptor for the capture of,

the method comprises the following steps:

loading a liquid comprising an analyte in the main microfluidic channel, advancing the liquid along the main microfluidic channel, through the intersection and interacting there with receptors for the captured microbeads.